Relationship between fertilizing ability of frozen human spermatozoa and capacity for heparin binding and nuclear decondensation
B. Lassalle and J. Testart INSERM, unité 335, 32
rue
des Carnets, F-92140 Clamart, France
Summary. Nuclear decondensation of spermatozoa induced by heparin, reduced glutathione (GSH) or a mixture of heparin and GSH was studied using frozen-
thawed human spermatozoa. The percentages of decondensed spermatozoa in controls and after treatment for 60 min with 30 \g=m\molheparin 1\m=-\1, 5 mmol GSH 1\m=-\1, or heparin\p=n-\GSH mixture were 1\m=.\5,22\m=.\1, 4\m=.\3 and 37\m=.\6%, respectively. Most of the decondensed spermatozoa were eliminated by Percoll gradient centrifugation of samples previously treated with heparin or heparin\p=n-\GSHmixture. However, comparable numbers of motile spermatozoa were recovered in the control and in each treated sample, demonstrating that a major proportion of motile spermatozoa was resistant to heparin (or heparin\p=n-\GSH)effects on nuclear decondensation of
spermatozoa.
Fertilization of hamster oocytes was attempted using spermatozoa recovered in the 90% Percoll fraction and resistant to heparin\p=n-\GSHdecondensing mixture. Although insemination used a constant number of motile spermatozoa, fertilization rates were higher after treatments with heparin and GSH alone than in control or heparin\p=n-\ GSH-treated samples. In addition the number of spermatozoa that attached to the oocyte plasma membrane was a sixth or a half for sperm pretreated with heparin\p=n-\GSH or heparin alone, respectively compared with untreated values. However, there was no evidence for induced acrosomal reaction by heparin and GSH, at least at the concentrations used. Qualitative analyses of heparin-binding sites were performed on untreated spermatozoa recovered in the 90% Percoll fraction by incubating spermatozoa in the presence of heparin covalently linked to albumin and coupled to colloidal gold (5 nm). Among this population of spermatozoa, 40\m=.\5%bound heparin\p=n-\goldand labelling was mainly observed on the sperm head surface (88% of labelled spermatozoa) with (59\m=.\5%)or without (28\m=.\5%)tail labelling. Only a small proportion (23%) of spermatozoa that attached to the oocyte plasma membrane bound heparin\p=n-\goldconjugate and only weak labelling was observed on the sperm head. Moreover, the proportion of spermatozoa that bound heparin\p=n-\goldconjugate decreased (r \m=-\0\m=.\77, P < 0\m=.\0001)in relation to increasing concentrations of motile spermatozoa in the =
sample.
Our results obtained with frozen\p=n-\thawedspermatozoa show that (i) there is a close relationship between sperm motility and the inability of spermatozoa to bind heparin, (ii) a high decondensing capacity of human spermatozoa is not representative of a higher fertilizing competence, and (iii) most motile spermatozoa do not bind heparin on
their membranes.
Keywords: heparin; glutathione; sperm nuclear decondensation; spermatozoa\p=n-\egginteraction; human
Introduction After sperm-egg fusion, the first events that permit a male contribution to the embryonic genome are nuclear decondensation of spermatozoa and male pronuclear formation. It has been postulated that adequate condensation and stability of sperm chromatin is essential to protect the genome from physical, chemical and mutagenic agents until the male gamete reaches the egg (Saowaros & Panyim, 1979; Bustos-Obregon & Leiva, 1983). An adequate decondensation of sperm chromatin is also necessary for male pronucleus formation after fertilization. Nuclear decondensation in spermatozoa has been induced in vitro by several chemical agents (reviewed by Huret, 1986). Among these agents, glycosaminoglycan (GAG) heparin, when used alone and at high nonphysiological concentrations (0-2-2 mmol l"1), was found to have specific decondensing ability in the human sperm nucleus (Delgado et ai, 1980). GAGs also stimulate the acrosome reaction in mammals (bulls: review by Miller & Ax, 1990; pigs: Reyes et ai, 1984; rabbits: Lenz et ai, 1983; hamsters: Meizel & Turner, 1986) and humans (Valencia et ai, 1984; Delgado et ai, 1988). Moreover, heparin binds to the membrane of human spermatozoa (Delgado et ai, 1982) probably at the sites of heparin-binding proteins of seminal plasma origin (Miller & Ax, 1990; Nass et ai, 1990) with a binding process analogous to that of cell surface receptors (Delgado et ai, 1982; Handrow et ai, 1984). Heparin may cause destabilization of the membrane of spermatozoa, ensuring an acrosomal reaction and internalization of GAGs and inducing the decondensation process in the sperm nucleus (Delgado et ai, 1982; Reyes et ai, 1984). Moreover, GAGs are present in the genital tracts of men (Sampaio et ai, 1985) and women (Lee & Ax, 1984) and in follicular fluid (Grimek et ai, 1984). A heparin-like GAG from bovine oviductal fluid may induce capacitation of bovine spermatozoa (Parrish et ai, 1989). Reduced glutathione (GSH), a tripeptide containing cysteine and the most abundant nonprotein thiol in several mammalian cells, maintains reducing ability in the cell (Jocelyn, 1972). A GSH concentration of 8 mmol l"1 per mature hamster oocyte has been estimated by Perreault et al. (1988) using enzymatic microassay. It has been suggested that GSH plays a role in nuclear decondensation of spermatozoa as a possible effector of disulphide bond reduction in sperm chromatin (reviewed by Zirkin et ai, 1985). When 31-6 pmol heparin 1" ' was associated with 5 mmol GSH 1" ' in sperm suspension (Reyes et ai, 1989) the same high proportion of decondensing spermatozoa was obtained (90%) as with 2000 pmol heparin l"1 alone (Delgado et ai, 1980). However, the time needed to obtain 90% decondensed spermatozoa was 1 and 6 h, respectively. Sulfhydryl compounds such as reduced glutathione (GSH) and dithiothreitol (DTT), even at high concentrations, cannot induce decon¬ densation of sperm nuclei (Kvist, 1980; Huret, 1986) except after addition either to detergents (Evenson et ai, 1978; Sobhon et ai, 1981; Bustos-Obregon & Leiva, 1983) or to other compounds (Gall & Ohsumi, 1976). The aim of this study was to induce nuclear decondensation of spermatozoa artificially and to evaluate the fertilizing ability of those human spermatozoa that were resistant to heparin or heparin-GSH decondensing agents. As motility and fertilizing ability of spermatozoa are lost if decondensation occurs before spermatozoa-oocyte interaction, Percoli gradient centrifugation was used to eliminate the population of decondensed spermatozoa and to recover intact spermatozoa. Fertilization was then assessed in the human-hamster heterospecific model.
Materials and Methods
Preparation of spermatozoa The same frozen ejaculate from donor A was used to study nuclear decondensation of spermatozoa and fertilizing ability of spermatozoa after various treatments. Frozen spermatozoa allowed the use of several straws for successive
assays and limited inter- and intra-individual variations of sperm parameters. Frozen semen from five donors, includ¬ ing donor A, permitted identification of heparin-binding sites and analysis of acrosome status in treated spermatozoa. The cryoprotectant medium and the freezing method used were as described by Jondet (1976). Spermatozoa were thawed at room temperature and the contents of four straws (0-25 ml each) were diluted with 5 ml Dulbecco's phosphate-buffered saline (PBS) plus 0-4% (w/v) bovine serum albumin (BSA, fraction V: Sigma, 38297 La Verpillière, France). The sperm suspension was centrifuged at 600 g for 7 min and the pellet was resuspended in 4 ml B2 medium (B2 Menezo: Bio Merieux SA, 69280 Marcy-l'Etoile, France).
Treatment of spermatozoa with
heparin, GSH and heparin-GSH mixture
Heparin (M, 13 000, 176 USP mg"1, H-3125; Sigma) stock solution (307-6pmol l"1 in B2) and reduced glutathione (GSH; Sigma) stock solution (50mmol l"1, pH 7-4) were prepared just before sperm treatment. Then, 100 µ of either heparin stock solution, GSH stock solution or both solutions were added to 0-8 ml sperm suspension. For heparin or GSH treatment, 100 µ B2 was added to obtain a constant final volume of 1 ml. The final concen¬ trations in the sperm suspension were either 30-1 µ heparin 1 ', 5 mmol GSH 1 ' and 30-1 pmol heparin-5 mmol GSH 1 '. The concentrations of heparin and GSH used in our study were considered as physiological according to the estimated concentrations of heparin sulfate (20-28 pmol l"1) in bovine follicular fluid (Bellin & Ax, 1987) or GSH (8 mmol 1" ') in hamster oocytes (Perreault et ai, 1988). Control samples were obtained by the addition of 200 µ B2 to 0-8 ml sperm suspension. Control and treated samples were incubated for 60 min in a C02 incubator (37°C, 5% C02). The proportion of nuclear decondensation of spermatozoa was estimated in each sample before centrifugation on discontinuous Percoli gradients prepared as follows: an isoosmotic solution containing 90% Percoli was obtained by mixing 9 volumes of Percoli (Pharmacia LKB, Uppsala, Sweden) with 1 volume of 10-fold concentrated Ham's FIO medium (Gibco, 95051 Cergy-Pontoise, France) medium. This 90% Percoli solution was used to prepare 70, 50 and 30% Percoli solutions by dilution with B2 medium and an increasing gradient was obtained by placing I ml of each solution gently in a conical culture tube, before addition of sperm sample. After centrifugation (for 15 min at 600 g), the 90% Percoli fraction was carefully recovered with a pipette, then diluted in PBS-BSA medium (5 ml) and centri¬ fuged at 600g for 7 min. Pellets were resuspended in 0-5 ml B2 medium to constitute the insemination medium and 100 µ sperm suspension was kept for sperm count and examination. sulfate
"
"
~
Collection of hamster oocytes and fertilization
Virgin golden hamsters were induced to superovulate by intraperitoneal injection of 40 iu pregnant mares' serum gonadotrophin (PMSG; Intervet, 49100 Angers, France), followed by 40 iu human chorionic gonadotrophin (hCG; Intervet) 72 h later. Animals were killed 15-17 h after hCG injection. Cumulus cells were dispersed with 01% hyaluronidase (bovine testis, type I: Sigma), and zonae pellucidae were removed by treatment with 005% trypsin (type I, Sigma) solution in PBS-BSA. Zona-free oocytes were thoroughly rinsed in fresh PBS-BSA medium after each enzymatic treatment and finally placed in B2 medium at 37°C under 5% C02. Concentration of spermatozoa in the 106 spermatozoa ml"'. Insemination medium was placed into macrowells insemination medium was 0-70-0-75 (400 µ per well) of Nunclon delta dishes (Nunc, Roskilde, Denmark) before addition of 13-26 zona-free oocytes and the culture was performed in a C02 incubator (37°C, 5% C02). Control of nuclear decondensation of spermatozoa was performed on each sample before Percoli centrifugation. Motile and nonmotile counted with the help of a Malassez cell (depth: 0-2 mm; area: 00025 mm2). The proportion of decondensed spermatozoa was assessed before Percoli gradient centrifugation by using either the method of Krzanowska (1982) with absolute ethanol: glacial acetic acid (3:1) fixation of sperm samples followed with toluidine blue staining or the method of Kvist ( 1980), slightly modified, in which nonfixed sperm samples were placed between a slide and a coverslip and observed under a phase contrast microscope ( 400). Sperm nuclei were considered 'decondensed' when sperm nuclear swelling was observed in comparison with the intact sperm nucleus (Fig. la, b). Percentages were calculated from at least 200 spermatozoa per preparation.
Analysis of spermatozoa
spermatozoa
were
Identification of heparin-binding sites
on
human spermatozoa
Qualitative analysis of heparin-binding sites on human spermatozoa was performed by phase-contrast microscopy ( 1000). A solution of spermatozoa (1 ml B2) was incubated for 30 min with heparin-albumin, gold-labelled (H 9516, 5 nm gold particle diameter, Sigma) at 1:500 dilution, then washed. The sperm pellet was resuspended in 0-5 ml glutaraldehyde (10%) for 5 min, then washed with distilled water and the final pellet spread and air-dried on a slide as for cytological preparation. Precipitation of metallic silver occurred from addition of silver enhancer solutions (Silver Enhancer Kit: Sigma) to the cytological preparation; this enlarged colloidal gold labels, yielding high-contrast signals visible by light microscopy. The preparation was finally fixed by immersing the slide in 2-5% aqueous sodium thiosulfate pentahydrate for 3 min, then rinsed in distilled water and a coverslip was mounted over Glycergel (Dako
Fig. 1. The proportion of decondensed spermatozoa assessed before Percoli gradient centri¬ fugation from acetic alcohol-fixed samples (a) stained with toluidine blue or (b) directly from fresh samples (unfixed) placed between slide and coverslip. Sperm nuclei were considered 'decondensed' when a visible nuclear swelling (arrow) was observed in comparison with an intact sperm nucleus. Percentages were calculated from at least 200 spermatozoa per
preparation. Phase contrast,
80.
SA, 78006 Versailles, France). Heparin-binding sites on the membrane of spermatozoa were identified when scattered
compacted dark grains were observed (Fig. 2a-g). Occasionally very compacted grains appeared réfringent with a gold metallic aspect (Fig. 2b). Four experiments were performed to determine the specificity of this procedure. Experiment 1. The appropriate concentration of conjugate was defined empirically by addition of various dilutions IO6 spermatozoa ml"1). An adequate dilution of heparin-gold of the commercial solution to sperm samples (1 conjugate was 1:500 of the commercial solution. Insufficient binding of heparin gold conjugate with consequent
or
2. Identification of heparin-binding sites on human spermatozoa was performed from untreated spermatozoa recovered in the 90% Percoli gradient. Heparin was coupled to colloi¬ dal gold (5 nm) and amplification occurred with the Sigma Silver Enhancer Kit. (a) Unlabelled (o) and heparin-gold labelled (right) spermatozoa. Dark grains (arrows) appear simul¬ taneously on the head anterior (scattered), equatorial region (dense) or tail, (b) Sperm head and midpiece entirely labelled but tail partly labelled, (c) Sperm head and tail partly labelled, (d) Sperm head labelling only, (e) Tail labelling only, (f) Heterogeneity of heparin-binding sites in a sperm population (o, no labelling; h, head labelling; t, tail labelling; h + t, head and tail labelling), (g) Identification of heparin-binding sites on the spermatozoa that attached to zonafree hamster oocytes. Labelling was observed on the head only and consisted of some dark grains (arrow) at the surface of sperm head. Phase-contrast, 80.
Fig.
difficulties of interpretation after sperm incubation with
observed between dilutions of 1:500 and 1:1000, whereas dilution beyond 1:1000.
was a
no
binding was observed
Experiment 2. Solutions of spermatozoa were treated with different concentrations of heparin (0, 30, 60, 150 and l"1) then washed twice and finally treated with heparin-gold. There was a concentration-dependent
300 µ
—0-99) of heparin-gold binding on the sperm surface, since 53-7, 31-3, 19-7, 17-7 and 101% of spermatozoa bound 'heparin-gold conjugate when solutions of spermatozoa were pretreated with 0, 30, 60, 150 and 300 pmol heparin 1 before the addition of heparin-gold conjugate, respectively. Experiment 3. Heparin-gold binding after pretreatment of solutions of spermatozoa with 300 µ heparin l"1 was inhibited with 1:500 but not with 1:160 or 1:250 dilutions of heparin-gold. decrease (r
=
"
Experiment 4. Sperm solutions were pretreated for 30 min with five glycoaminoglycans including heparin, then finally treated with gold labelled heparin-albumin at the concentration determined above. Only heparin and dextran sulfate (Sigma, Mr 5000; 800 µ l"1) showed significant inhibition of heparin-gold l"1) (30 pmol binding on spermatozoa. Dextran sulfate was reported to be a competitive agent of heparin (Delgado et at, 1982). However, no inhibition was observed after treatment with chondroitin sulfate A and (Sigma, Mr 50 000; 80 µ l"1) and hyaluronic acid (4 mg ml"1). These results were comparable to those of Delgado et ai (1982) using washed and
radioactive GAGs. Identification of heparin-binding sites on spermatozoa bound to zona-free hamster oocytes was assessed after insemination with washed sperm not submitted to Percoli selection but suspended in 1 ml B2 medium (Lassalle & Testart, 1989). Heparin-gold conjugate was added 30 min before insemination with the sperm sample and remained present during sperm-egg interaction. Three hours after insemination, eggs were thoroughly washed, then fixed (45 min) with 3% (w/v) paraformaldehyde in PBS, washed twice in distilled water and air-dried on a slide. The Silver Enhancer Kit procedure was performed as described above and a coverslip was mounted over Glycergel.
Evaluation of viability and acrosome status of spermatozoa treated with heparin The double-staining technique using single-wavelength fluorescence microscopy was performed to evaluate the viability and acrosome status of spermatozoa as previously described (Centola et ai, 1990). Sperm cells were stained with propidium iodide (PI) in combination with fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA) which binds to the acrosome content of permeabilized acrosome-intact spermatozoa (Cross et at, 1986). At least 100 sperm cells were counted for each specimen.
Control of fertilization (
Zona-free oocytes were placed between slide and coverslip for observation under a phase contrast microscope 1000) 3-4 h after insemination. Ova were recorded as penetrated or polyspermic when at least one (penetrated) or
(polyspermic) male pronuclei or decondensed sperm nuclei were discernible in the egg ooplasm. Results were expressed according to different ratios: (1) penetration rate (percentage of penetrated oocytes); (2) polyspermic oocyte rate (average number of polyspermic oocytes among penetrated oocytes). two
Statistical Data
analysis
were
expressed as
±sem and
compared using one-way analysis of variance (anova).
Results The percentages of decondensed spermatozoa estimated before Percoli gradient centrifugation increased in samples treated with heparin-GSH (37-6%) and heparin (22-1%) compared to GSH-treated (4-3%) and control (15%) spermatozoa (Table 1). In samples that were fixed by acetic alcohol the corresponding results were 6-5, 560, 70-5 and 570%, respectively. However, the percentage of decondensed pretreated spermatozoa in the 90% Percoli fraction was very low (1-2%) and similar to that of untreated spermatozoa, whereas, in the 70 or 50% Percoli fractions, decondensed and free tail spermatozoa were observed in high proportions. The concentration of spermatozoa recovered in the 90% Percoli fraction decreased in samples pretreated with heparin-GSH (21 106 ml"1) or heparin (4-6 106 ml"1) compared with 106 ml"1) samples (Table 1). In addition control (7-4 106 ml"1) and GSH-pretreated (71 the total number of spermatozoa recovered decreased when the rate of decondensed spermatozoa before centrifugation increased (r —0-99, < 0001). However, comparable numbers of motile spermatozoa were recovered from control and pretreated samples (Table 1 ). As the number of motile spermatozoa was not modified by the treatments, fertilization was assayed using a constant initial 106 ml"1) then pretreated or untreated and submitted number of motile spermatozoa (0-70-0-75 to Percoli gradient centrifugation. Penetrated oocyte rate increased using spermatozoa pretreated =
Table 1. Effects of
heparin,
reduced glutathione (GSH) and heparin-GSH mixture nuclear decondensation and motility Treatment of spermatozoa before Percoli
on
human sperm
gradient centrifugation
None
5 mmol
(control)
GSH1"1
heparin 1"
30 µ heparin 1 ' and 5 mmol GSH l"1
Concentration of motile spermatozoa
1-5 0-75
4-3 0-70
22-1 0-75
37-6 0-70
Total sperm concentration
7-4
7-1
4-6
2-1
Percentage of decondensed spermatozoa2
(xlO^r1)"
30 µ
(xlO^l"1)"
aEstimated before Percoli gradient centrifugation. bAfter Percoli gradient centrifugation and just before insemination.
with either heparin or GSH compared with control spermatozoa ( < 005) or spermatozoa pre¬ treated with heparin-GSH (P < 001), respectively (Table 2). However, no significant differences were observed between penetrated oocyte rate in control (870%) and heparin-GSH-pretreated (74-2%) spermatozoa (Table 2). Polyspermic oocyte rate decreased when spermatozoa were pre¬ treated with heparin-GSH mixture compared with control and GSH-pretreated spermatozoa (P < 0001). Moreover, the rate of polyspermy was correlated (r = 0-99, < 001) with the number of spermatozoa bound per egg. Heparin pretreatment resulted in fewer spermatozoa bound per egg (46-8) compared with control (101-8, < 0001) or GSH-pretreated spermato¬ zoa (103-1, < 0001). Association of GSH with heparin still reduced the number of spermatozoa bound per egg (16-8 bound spermatozoa per egg) compared with control or single sperm pretreat¬ ment ( < 0001) (Table 2). However, the proportions of living acrosome-reacted spermatozoa were similar after treatment of spermatozoa with either heparin (43-8%), GSH (34-9%) or heparin-GSH mixture (29-1%) compared with the control (45-8%). Table 2. Penetration rate of hamster oocytes by spermatozoa different treatments
(0-70-0-75
106 ml"1) submitted
to
Pretreatment of spermatozoa
Penetrated oocyte
(%) Polyspermic oocyte (%)
Mean number of spermatozoa bound per egg ( + sem)
Results
ihP
were
obtained from
a
5 mmol
GSH1"1
870 (46)a-b 67-5 (40)e
101-8 ± 8-5f
100-0 67-9
(28)'·' (28)
103-1 + 7-l8h
30 pmol heparin 1" 100-0 48-2
(27)bd (27)
46-8 + 3-lM
30 pmol heparin 1 and 5 mmol GSH 74-2 28-6
(66)c-d (49)e
16-6 + l-5f·8·'
single donor ejaculate.
001; ef-8hi/> < 0001. Number of hamster oocytes analysed is given in parentheses.
B. Lassalle and J. Testart INSERM, unité 335, 32
rue
des Carnets, F-92140 Clamart, France
Summary. Nuclear decondensation of spermatozoa induced by heparin, reduced glutathione (GSH) or a mixture of heparin and GSH was studied using frozen-
thawed human spermatozoa. The percentages of decondensed spermatozoa in controls and after treatment for 60 min with 30 \g=m\molheparin 1\m=-\1, 5 mmol GSH 1\m=-\1, or heparin\p=n-\GSH mixture were 1\m=.\5,22\m=.\1, 4\m=.\3 and 37\m=.\6%, respectively. Most of the decondensed spermatozoa were eliminated by Percoll gradient centrifugation of samples previously treated with heparin or heparin\p=n-\GSHmixture. However, comparable numbers of motile spermatozoa were recovered in the control and in each treated sample, demonstrating that a major proportion of motile spermatozoa was resistant to heparin (or heparin\p=n-\GSH)effects on nuclear decondensation of
spermatozoa.
Fertilization of hamster oocytes was attempted using spermatozoa recovered in the 90% Percoll fraction and resistant to heparin\p=n-\GSHdecondensing mixture. Although insemination used a constant number of motile spermatozoa, fertilization rates were higher after treatments with heparin and GSH alone than in control or heparin\p=n-\ GSH-treated samples. In addition the number of spermatozoa that attached to the oocyte plasma membrane was a sixth or a half for sperm pretreated with heparin\p=n-\GSH or heparin alone, respectively compared with untreated values. However, there was no evidence for induced acrosomal reaction by heparin and GSH, at least at the concentrations used. Qualitative analyses of heparin-binding sites were performed on untreated spermatozoa recovered in the 90% Percoll fraction by incubating spermatozoa in the presence of heparin covalently linked to albumin and coupled to colloidal gold (5 nm). Among this population of spermatozoa, 40\m=.\5%bound heparin\p=n-\goldand labelling was mainly observed on the sperm head surface (88% of labelled spermatozoa) with (59\m=.\5%)or without (28\m=.\5%)tail labelling. Only a small proportion (23%) of spermatozoa that attached to the oocyte plasma membrane bound heparin\p=n-\goldconjugate and only weak labelling was observed on the sperm head. Moreover, the proportion of spermatozoa that bound heparin\p=n-\goldconjugate decreased (r \m=-\0\m=.\77, P < 0\m=.\0001)in relation to increasing concentrations of motile spermatozoa in the =
sample.
Our results obtained with frozen\p=n-\thawedspermatozoa show that (i) there is a close relationship between sperm motility and the inability of spermatozoa to bind heparin, (ii) a high decondensing capacity of human spermatozoa is not representative of a higher fertilizing competence, and (iii) most motile spermatozoa do not bind heparin on
their membranes.
Keywords: heparin; glutathione; sperm nuclear decondensation; spermatozoa\p=n-\egginteraction; human
Introduction After sperm-egg fusion, the first events that permit a male contribution to the embryonic genome are nuclear decondensation of spermatozoa and male pronuclear formation. It has been postulated that adequate condensation and stability of sperm chromatin is essential to protect the genome from physical, chemical and mutagenic agents until the male gamete reaches the egg (Saowaros & Panyim, 1979; Bustos-Obregon & Leiva, 1983). An adequate decondensation of sperm chromatin is also necessary for male pronucleus formation after fertilization. Nuclear decondensation in spermatozoa has been induced in vitro by several chemical agents (reviewed by Huret, 1986). Among these agents, glycosaminoglycan (GAG) heparin, when used alone and at high nonphysiological concentrations (0-2-2 mmol l"1), was found to have specific decondensing ability in the human sperm nucleus (Delgado et ai, 1980). GAGs also stimulate the acrosome reaction in mammals (bulls: review by Miller & Ax, 1990; pigs: Reyes et ai, 1984; rabbits: Lenz et ai, 1983; hamsters: Meizel & Turner, 1986) and humans (Valencia et ai, 1984; Delgado et ai, 1988). Moreover, heparin binds to the membrane of human spermatozoa (Delgado et ai, 1982) probably at the sites of heparin-binding proteins of seminal plasma origin (Miller & Ax, 1990; Nass et ai, 1990) with a binding process analogous to that of cell surface receptors (Delgado et ai, 1982; Handrow et ai, 1984). Heparin may cause destabilization of the membrane of spermatozoa, ensuring an acrosomal reaction and internalization of GAGs and inducing the decondensation process in the sperm nucleus (Delgado et ai, 1982; Reyes et ai, 1984). Moreover, GAGs are present in the genital tracts of men (Sampaio et ai, 1985) and women (Lee & Ax, 1984) and in follicular fluid (Grimek et ai, 1984). A heparin-like GAG from bovine oviductal fluid may induce capacitation of bovine spermatozoa (Parrish et ai, 1989). Reduced glutathione (GSH), a tripeptide containing cysteine and the most abundant nonprotein thiol in several mammalian cells, maintains reducing ability in the cell (Jocelyn, 1972). A GSH concentration of 8 mmol l"1 per mature hamster oocyte has been estimated by Perreault et al. (1988) using enzymatic microassay. It has been suggested that GSH plays a role in nuclear decondensation of spermatozoa as a possible effector of disulphide bond reduction in sperm chromatin (reviewed by Zirkin et ai, 1985). When 31-6 pmol heparin 1" ' was associated with 5 mmol GSH 1" ' in sperm suspension (Reyes et ai, 1989) the same high proportion of decondensing spermatozoa was obtained (90%) as with 2000 pmol heparin l"1 alone (Delgado et ai, 1980). However, the time needed to obtain 90% decondensed spermatozoa was 1 and 6 h, respectively. Sulfhydryl compounds such as reduced glutathione (GSH) and dithiothreitol (DTT), even at high concentrations, cannot induce decon¬ densation of sperm nuclei (Kvist, 1980; Huret, 1986) except after addition either to detergents (Evenson et ai, 1978; Sobhon et ai, 1981; Bustos-Obregon & Leiva, 1983) or to other compounds (Gall & Ohsumi, 1976). The aim of this study was to induce nuclear decondensation of spermatozoa artificially and to evaluate the fertilizing ability of those human spermatozoa that were resistant to heparin or heparin-GSH decondensing agents. As motility and fertilizing ability of spermatozoa are lost if decondensation occurs before spermatozoa-oocyte interaction, Percoli gradient centrifugation was used to eliminate the population of decondensed spermatozoa and to recover intact spermatozoa. Fertilization was then assessed in the human-hamster heterospecific model.
Materials and Methods
Preparation of spermatozoa The same frozen ejaculate from donor A was used to study nuclear decondensation of spermatozoa and fertilizing ability of spermatozoa after various treatments. Frozen spermatozoa allowed the use of several straws for successive
assays and limited inter- and intra-individual variations of sperm parameters. Frozen semen from five donors, includ¬ ing donor A, permitted identification of heparin-binding sites and analysis of acrosome status in treated spermatozoa. The cryoprotectant medium and the freezing method used were as described by Jondet (1976). Spermatozoa were thawed at room temperature and the contents of four straws (0-25 ml each) were diluted with 5 ml Dulbecco's phosphate-buffered saline (PBS) plus 0-4% (w/v) bovine serum albumin (BSA, fraction V: Sigma, 38297 La Verpillière, France). The sperm suspension was centrifuged at 600 g for 7 min and the pellet was resuspended in 4 ml B2 medium (B2 Menezo: Bio Merieux SA, 69280 Marcy-l'Etoile, France).
Treatment of spermatozoa with
heparin, GSH and heparin-GSH mixture
Heparin (M, 13 000, 176 USP mg"1, H-3125; Sigma) stock solution (307-6pmol l"1 in B2) and reduced glutathione (GSH; Sigma) stock solution (50mmol l"1, pH 7-4) were prepared just before sperm treatment. Then, 100 µ of either heparin stock solution, GSH stock solution or both solutions were added to 0-8 ml sperm suspension. For heparin or GSH treatment, 100 µ B2 was added to obtain a constant final volume of 1 ml. The final concen¬ trations in the sperm suspension were either 30-1 µ heparin 1 ', 5 mmol GSH 1 ' and 30-1 pmol heparin-5 mmol GSH 1 '. The concentrations of heparin and GSH used in our study were considered as physiological according to the estimated concentrations of heparin sulfate (20-28 pmol l"1) in bovine follicular fluid (Bellin & Ax, 1987) or GSH (8 mmol 1" ') in hamster oocytes (Perreault et ai, 1988). Control samples were obtained by the addition of 200 µ B2 to 0-8 ml sperm suspension. Control and treated samples were incubated for 60 min in a C02 incubator (37°C, 5% C02). The proportion of nuclear decondensation of spermatozoa was estimated in each sample before centrifugation on discontinuous Percoli gradients prepared as follows: an isoosmotic solution containing 90% Percoli was obtained by mixing 9 volumes of Percoli (Pharmacia LKB, Uppsala, Sweden) with 1 volume of 10-fold concentrated Ham's FIO medium (Gibco, 95051 Cergy-Pontoise, France) medium. This 90% Percoli solution was used to prepare 70, 50 and 30% Percoli solutions by dilution with B2 medium and an increasing gradient was obtained by placing I ml of each solution gently in a conical culture tube, before addition of sperm sample. After centrifugation (for 15 min at 600 g), the 90% Percoli fraction was carefully recovered with a pipette, then diluted in PBS-BSA medium (5 ml) and centri¬ fuged at 600g for 7 min. Pellets were resuspended in 0-5 ml B2 medium to constitute the insemination medium and 100 µ sperm suspension was kept for sperm count and examination. sulfate
"
"
~
Collection of hamster oocytes and fertilization
Virgin golden hamsters were induced to superovulate by intraperitoneal injection of 40 iu pregnant mares' serum gonadotrophin (PMSG; Intervet, 49100 Angers, France), followed by 40 iu human chorionic gonadotrophin (hCG; Intervet) 72 h later. Animals were killed 15-17 h after hCG injection. Cumulus cells were dispersed with 01% hyaluronidase (bovine testis, type I: Sigma), and zonae pellucidae were removed by treatment with 005% trypsin (type I, Sigma) solution in PBS-BSA. Zona-free oocytes were thoroughly rinsed in fresh PBS-BSA medium after each enzymatic treatment and finally placed in B2 medium at 37°C under 5% C02. Concentration of spermatozoa in the 106 spermatozoa ml"'. Insemination medium was placed into macrowells insemination medium was 0-70-0-75 (400 µ per well) of Nunclon delta dishes (Nunc, Roskilde, Denmark) before addition of 13-26 zona-free oocytes and the culture was performed in a C02 incubator (37°C, 5% C02). Control of nuclear decondensation of spermatozoa was performed on each sample before Percoli centrifugation. Motile and nonmotile counted with the help of a Malassez cell (depth: 0-2 mm; area: 00025 mm2). The proportion of decondensed spermatozoa was assessed before Percoli gradient centrifugation by using either the method of Krzanowska (1982) with absolute ethanol: glacial acetic acid (3:1) fixation of sperm samples followed with toluidine blue staining or the method of Kvist ( 1980), slightly modified, in which nonfixed sperm samples were placed between a slide and a coverslip and observed under a phase contrast microscope ( 400). Sperm nuclei were considered 'decondensed' when sperm nuclear swelling was observed in comparison with the intact sperm nucleus (Fig. la, b). Percentages were calculated from at least 200 spermatozoa per preparation.
Analysis of spermatozoa
spermatozoa
were
Identification of heparin-binding sites
on
human spermatozoa
Qualitative analysis of heparin-binding sites on human spermatozoa was performed by phase-contrast microscopy ( 1000). A solution of spermatozoa (1 ml B2) was incubated for 30 min with heparin-albumin, gold-labelled (H 9516, 5 nm gold particle diameter, Sigma) at 1:500 dilution, then washed. The sperm pellet was resuspended in 0-5 ml glutaraldehyde (10%) for 5 min, then washed with distilled water and the final pellet spread and air-dried on a slide as for cytological preparation. Precipitation of metallic silver occurred from addition of silver enhancer solutions (Silver Enhancer Kit: Sigma) to the cytological preparation; this enlarged colloidal gold labels, yielding high-contrast signals visible by light microscopy. The preparation was finally fixed by immersing the slide in 2-5% aqueous sodium thiosulfate pentahydrate for 3 min, then rinsed in distilled water and a coverslip was mounted over Glycergel (Dako
Fig. 1. The proportion of decondensed spermatozoa assessed before Percoli gradient centri¬ fugation from acetic alcohol-fixed samples (a) stained with toluidine blue or (b) directly from fresh samples (unfixed) placed between slide and coverslip. Sperm nuclei were considered 'decondensed' when a visible nuclear swelling (arrow) was observed in comparison with an intact sperm nucleus. Percentages were calculated from at least 200 spermatozoa per
preparation. Phase contrast,
80.
SA, 78006 Versailles, France). Heparin-binding sites on the membrane of spermatozoa were identified when scattered
compacted dark grains were observed (Fig. 2a-g). Occasionally very compacted grains appeared réfringent with a gold metallic aspect (Fig. 2b). Four experiments were performed to determine the specificity of this procedure. Experiment 1. The appropriate concentration of conjugate was defined empirically by addition of various dilutions IO6 spermatozoa ml"1). An adequate dilution of heparin-gold of the commercial solution to sperm samples (1 conjugate was 1:500 of the commercial solution. Insufficient binding of heparin gold conjugate with consequent
or
2. Identification of heparin-binding sites on human spermatozoa was performed from untreated spermatozoa recovered in the 90% Percoli gradient. Heparin was coupled to colloi¬ dal gold (5 nm) and amplification occurred with the Sigma Silver Enhancer Kit. (a) Unlabelled (o) and heparin-gold labelled (right) spermatozoa. Dark grains (arrows) appear simul¬ taneously on the head anterior (scattered), equatorial region (dense) or tail, (b) Sperm head and midpiece entirely labelled but tail partly labelled, (c) Sperm head and tail partly labelled, (d) Sperm head labelling only, (e) Tail labelling only, (f) Heterogeneity of heparin-binding sites in a sperm population (o, no labelling; h, head labelling; t, tail labelling; h + t, head and tail labelling), (g) Identification of heparin-binding sites on the spermatozoa that attached to zonafree hamster oocytes. Labelling was observed on the head only and consisted of some dark grains (arrow) at the surface of sperm head. Phase-contrast, 80.
Fig.
difficulties of interpretation after sperm incubation with
observed between dilutions of 1:500 and 1:1000, whereas dilution beyond 1:1000.
was a
no
binding was observed
Experiment 2. Solutions of spermatozoa were treated with different concentrations of heparin (0, 30, 60, 150 and l"1) then washed twice and finally treated with heparin-gold. There was a concentration-dependent
300 µ
—0-99) of heparin-gold binding on the sperm surface, since 53-7, 31-3, 19-7, 17-7 and 101% of spermatozoa bound 'heparin-gold conjugate when solutions of spermatozoa were pretreated with 0, 30, 60, 150 and 300 pmol heparin 1 before the addition of heparin-gold conjugate, respectively. Experiment 3. Heparin-gold binding after pretreatment of solutions of spermatozoa with 300 µ heparin l"1 was inhibited with 1:500 but not with 1:160 or 1:250 dilutions of heparin-gold. decrease (r
=
"
Experiment 4. Sperm solutions were pretreated for 30 min with five glycoaminoglycans including heparin, then finally treated with gold labelled heparin-albumin at the concentration determined above. Only heparin and dextran sulfate (Sigma, Mr 5000; 800 µ l"1) showed significant inhibition of heparin-gold l"1) (30 pmol binding on spermatozoa. Dextran sulfate was reported to be a competitive agent of heparin (Delgado et at, 1982). However, no inhibition was observed after treatment with chondroitin sulfate A and (Sigma, Mr 50 000; 80 µ l"1) and hyaluronic acid (4 mg ml"1). These results were comparable to those of Delgado et ai (1982) using washed and
radioactive GAGs. Identification of heparin-binding sites on spermatozoa bound to zona-free hamster oocytes was assessed after insemination with washed sperm not submitted to Percoli selection but suspended in 1 ml B2 medium (Lassalle & Testart, 1989). Heparin-gold conjugate was added 30 min before insemination with the sperm sample and remained present during sperm-egg interaction. Three hours after insemination, eggs were thoroughly washed, then fixed (45 min) with 3% (w/v) paraformaldehyde in PBS, washed twice in distilled water and air-dried on a slide. The Silver Enhancer Kit procedure was performed as described above and a coverslip was mounted over Glycergel.
Evaluation of viability and acrosome status of spermatozoa treated with heparin The double-staining technique using single-wavelength fluorescence microscopy was performed to evaluate the viability and acrosome status of spermatozoa as previously described (Centola et ai, 1990). Sperm cells were stained with propidium iodide (PI) in combination with fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin (PSA) which binds to the acrosome content of permeabilized acrosome-intact spermatozoa (Cross et at, 1986). At least 100 sperm cells were counted for each specimen.
Control of fertilization (
Zona-free oocytes were placed between slide and coverslip for observation under a phase contrast microscope 1000) 3-4 h after insemination. Ova were recorded as penetrated or polyspermic when at least one (penetrated) or
(polyspermic) male pronuclei or decondensed sperm nuclei were discernible in the egg ooplasm. Results were expressed according to different ratios: (1) penetration rate (percentage of penetrated oocytes); (2) polyspermic oocyte rate (average number of polyspermic oocytes among penetrated oocytes). two
Statistical Data
analysis
were
expressed as
±sem and
compared using one-way analysis of variance (anova).
Results The percentages of decondensed spermatozoa estimated before Percoli gradient centrifugation increased in samples treated with heparin-GSH (37-6%) and heparin (22-1%) compared to GSH-treated (4-3%) and control (15%) spermatozoa (Table 1). In samples that were fixed by acetic alcohol the corresponding results were 6-5, 560, 70-5 and 570%, respectively. However, the percentage of decondensed pretreated spermatozoa in the 90% Percoli fraction was very low (1-2%) and similar to that of untreated spermatozoa, whereas, in the 70 or 50% Percoli fractions, decondensed and free tail spermatozoa were observed in high proportions. The concentration of spermatozoa recovered in the 90% Percoli fraction decreased in samples pretreated with heparin-GSH (21 106 ml"1) or heparin (4-6 106 ml"1) compared with 106 ml"1) samples (Table 1). In addition control (7-4 106 ml"1) and GSH-pretreated (71 the total number of spermatozoa recovered decreased when the rate of decondensed spermatozoa before centrifugation increased (r —0-99, < 0001). However, comparable numbers of motile spermatozoa were recovered from control and pretreated samples (Table 1 ). As the number of motile spermatozoa was not modified by the treatments, fertilization was assayed using a constant initial 106 ml"1) then pretreated or untreated and submitted number of motile spermatozoa (0-70-0-75 to Percoli gradient centrifugation. Penetrated oocyte rate increased using spermatozoa pretreated =
Table 1. Effects of
heparin,
reduced glutathione (GSH) and heparin-GSH mixture nuclear decondensation and motility Treatment of spermatozoa before Percoli
on
human sperm
gradient centrifugation
None
5 mmol
(control)
GSH1"1
heparin 1"
30 µ heparin 1 ' and 5 mmol GSH l"1
Concentration of motile spermatozoa
1-5 0-75
4-3 0-70
22-1 0-75
37-6 0-70
Total sperm concentration
7-4
7-1
4-6
2-1
Percentage of decondensed spermatozoa2
(xlO^r1)"
30 µ
(xlO^l"1)"
aEstimated before Percoli gradient centrifugation. bAfter Percoli gradient centrifugation and just before insemination.
with either heparin or GSH compared with control spermatozoa ( < 005) or spermatozoa pre¬ treated with heparin-GSH (P < 001), respectively (Table 2). However, no significant differences were observed between penetrated oocyte rate in control (870%) and heparin-GSH-pretreated (74-2%) spermatozoa (Table 2). Polyspermic oocyte rate decreased when spermatozoa were pre¬ treated with heparin-GSH mixture compared with control and GSH-pretreated spermatozoa (P < 0001). Moreover, the rate of polyspermy was correlated (r = 0-99, < 001) with the number of spermatozoa bound per egg. Heparin pretreatment resulted in fewer spermatozoa bound per egg (46-8) compared with control (101-8, < 0001) or GSH-pretreated spermato¬ zoa (103-1, < 0001). Association of GSH with heparin still reduced the number of spermatozoa bound per egg (16-8 bound spermatozoa per egg) compared with control or single sperm pretreat¬ ment ( < 0001) (Table 2). However, the proportions of living acrosome-reacted spermatozoa were similar after treatment of spermatozoa with either heparin (43-8%), GSH (34-9%) or heparin-GSH mixture (29-1%) compared with the control (45-8%). Table 2. Penetration rate of hamster oocytes by spermatozoa different treatments
(0-70-0-75
106 ml"1) submitted
to
Pretreatment of spermatozoa
Penetrated oocyte
(%) Polyspermic oocyte (%)
Mean number of spermatozoa bound per egg ( + sem)
Results
ihP
were
obtained from
a
5 mmol
GSH1"1
870 (46)a-b 67-5 (40)e
101-8 ± 8-5f
100-0 67-9
(28)'·' (28)
103-1 + 7-l8h
30 pmol heparin 1" 100-0 48-2
(27)bd (27)
46-8 + 3-lM
30 pmol heparin 1 and 5 mmol GSH 74-2 28-6
(66)c-d (49)e
16-6 + l-5f·8·'
single donor ejaculate.
001; ef-8hi/> < 0001. Number of hamster oocytes analysed is given in parentheses.
